The present invention relates to methods for analysis of glucose in biological fluids, especially blood plasma or serum, or in aerated urine. More particularly this invention relates to methods for regenerating the glucose oxidase reagent used in instrumental analyzers for determining glucose concentration, such as the Beckman Glucose Analyzer. Such analyzers are discussed in U.S. Pat. No. 3,765,841, column 2, line 44 ff.
In the Beckman Glucose Analyzer, glucose is determined by treating the sample containing glucose with an aerated enzyme reagent containing glucose oxidase and measuring the rate of resultant oxygen consumption. The oxygen content is measured by a sensor which responds to oxygen concentration. Solid-state electronic circuitry determines the rate of oxygen consumption, which is directly proportional to the concentration of glucose in the sample. The latter is indicated on the analyzer by direct readout in milligrams of glucose per 100 ml. of sample.
The Beckman glucose oxidase reagent consists of 150EU/ml of glucose oxidase (Aspergillus niger) in 5% denatured ethanol 10.sup.-.sup.2 M potassium iodide solution. The reagent also contains catalase, which occurs as a natural impurity in glucose oxidase, and ammonium molybdate as catalytic reagents prepared in a phosphate buffer with 5 .times. 10.sup.-.sup.4 iodine as a preservative.
When a biological sample containing glucose is injected into the aerated enzyme reagent solution, .beta.-D-glucose from the sample combines with dissolved oxygen from the solution according to the reaction: ##EQU1## In the above reaction the oxygen is consumed at the same rate as the glucose reacts.
Because oxygen consumption rather than peroxide formation is measured, the only requirement for peroxide is that it must be destroyed by a path not leading back to oxygen. The ethanol present in the reagent causes the H.sub.2 O.sub.2 to be destroyed by catalase, an impurity in the glucose oxidase, without yielding oxygen in accordance with the reaction: ##EQU2## To ensure destruction of the peroxide, iodide and molybdate are added to the enzyme reagent, causing the reaction ##EQU3## This reaction is effective even after catalase activity has diminished with storage.
Before analyses are performed in the Beckman Glucose Analyzer, the enzyme reagent must be aerated by shaking in the bottle several times with fresh portions of air. The aeration is necessary in order to provide the oxygen to react with the glucose according to equation (1).
A bottle of 250 ml. of enzyme reagent is used in the glucose analyzer and is sufficient for 250 glucose tests. In other words, 1 ml. of the enzyme reagent is conveyed to a sample cup for each test to be performed. The amount of sample analyzed in each test is 10 .mu.1. At the conclusion of each test, the contents of the sample cup are drained into a waste bottle. When all of the used enzyme reagent (including the tested samples) has been collected in the waste bottle, it is discarded and a new bottle of enzyme reagent placed in the analyzer.